The bioelectrical interactions and activity believed to be present in a variety of biological tissues and cells are one of the least understood of the physiological processes. However, there has recently been much research into these interactions and activity regarding the growth and repair of certain tissues and cells. In particular, there has been much research into stimulation by electric and electromagnetic fields and its effect on the growth and repair of bone, cartilage, and various growth factors. Researchers believe that such research may be useful in the development of new treatments for a variety of medical problems.
Fibroblastic growth factor-2 (FGF-2) is one of the growth factors that is important in promotion of bone formation and maintenance, from proliferation of pre-osteoblasts, to differentiation of pre-osteoblasts to mature osteoblasts, and to maintenance of the osteoblast throughout its life span. FGF-2 has been shown to have a positive anabolic effect on bone formation in intact animals and to reduce bone loss in experimental models of osteoporosis (Fromigue, O., Modrowski, D., and Marie, P. J: Curr. Pharmaceut. Design, 10: 2593-2603, 2004.). FGF-2 (also termed basic fibroblastic growth or bFGF) stimulates bone formation, decreases osteoclast surface, and induces new bone spicules within the marrow cavity of the tibia in ovariectomized rats (Liang H., Pun S., and Wronski, T. J.: Endocrinology, 140: 5780-88, 1999). FGF-2 has a strong stimulatory effect on new bone formation in ovariectomized rats by inducing the conversion of bone lining cells to osteoblasts (Power, R. A., Iwaniec, U. T., Magee, K. A., Mitova-Caneva, N. G., and Wronski, T. J.: Osteoporosis Int., 15: 716-23, 2004). FGF-2 increases peak bone mass in a murine model of low turnover osteoporosis by stimulating osteoprogenitor cells to proliferate and differentiate into osteoblasts, thereby enhancing endocortical bone remodeling (Nagai, H., Tsukuda, H., and Mayahara, H.: J. Vet. Med. Sci., 61: 869-75, 1999). In another study, FGF-2 improved bone mechanical properties (maximum force and work to failure) and increased the number, thickness, and connections of trabeculae in a small animal osteoporosis model (Yao, W., Hadi, T., Jiang, Y., Lotz, J., Wronski, T. J., and Lane, N. E.: Osteoporosis Int., 16: 1939-47, 2005).
Thus, up-regulation of FGF-2 may be useful in the treatment of the disease commonly known as osteoporosis, where bone demineralizes and becomes abnormally rarefied. Bone comprises an organic component of cells and matrix as well as an inorganic or mineral component. The cells and matrix comprise a framework of collagenous fibers that is impregnated with the mineral component of calcium phosphate (85%) and calcium carbonate (10%) that imparts rigidity to the bone. In healthy bone, bone formation and bone resorption are in balance. In osteoporosis, bone resorption exceeds bone formation, leading to bone weakening and possible vertebral body fracture and collapse. While osteoporosis is generally thought as afflicting the elderly, certain types of osteoporosis may affect persons of all ages whose bones are not subject to functional stress. In such cases, patients may experience a significant loss of cortical and cancellous bone during prolonged periods of immobilization. Elderly patients are known to experience bone loss due to disuse when immobilized after fracture of a bone, and such bone loss may ultimately lead to a secondary fracture in an already osteoporotic skeleton. Diminished bone density may lead not only to vertebrae collapse, but also to fractures of hips, lower arms, wrists, ankles as well as incapacitating pains. Alternative non-surgical therapies for such diseases are needed.
Pulsed electromagnetic fields (PEMF) and capacitive coupling (CC) have been used widely to treat nonhealing fractures (nonunion) and related problems in bone healing since approval by the Food and Drug Administration in 1979. The original basis for the trial of this form of therapy was the observation that physical stress on bone causes the appearance of tiny electric currents that, along with mechanical strain, were thought to be the mechanisms underlying transduction of the physical stresses into a signal that promotes bone formation. Along with direct electric field stimulation that was successful in the treatment of nonunion, noninvasive technologies using PEMF and capacitive coupling (where the electrodes are placed on the skin in the treatment zone) were also found to be effective. PEMFs generate small, induced currents (Faraday currents) in the highly-conductive extracellular fluid, while capacitive coupling directly causes currents in the tissues; both PEMFs and CC thereby mimic endogenous electrical currents.
The endogenous electrical currents, originally thought to be due to phenomena occurring at the surface of crystals in the bone, have been shown to be due primarily to movement of fluid containing electrolytes in channels of the bone containing organic constituents with fixed negative charges, generating what are called “streaming potentials.” Studies of electrical phenomena in bone have demonstrated a mechanical-electrical transduction mechanism that appears when bone is mechanically compressed, causing movement of fluid and electrolytes over the surface of fixed negative charges on the surface of bone cells, thus producing streaming potentials. These electrical potentials serve a purpose in bone, and, along with mechanical strain, lead to signal transduction that is capable of stimulating bone cell synthesis of a calcifiable matrix, and, hence, the formation of bone.
The main application of direct current, capacitive coupling, and PEMFs has been in orthopedics in healing of nonunion bone fractures (Brighton et al., J. Bone Joint Surg. 63: 2-13, 1981; Brighton and Pollack, J. Bone Joint Surg. 67: 577-585, 1985; Bassett et al., Crit. Rev. Biomed. Eng. 17: 451-529, 1989; Bassett et al., J. Am. Med. Assoc. 247: 623-628, 1982). Clinical responses have been reported in avascular necrosis of hips in adults and Legg-Perthes's disease in children (Bassett et al., Clin. Orthop. 246: 172-176, 1989; Aaron et al., Clin. Orthop. 249: 209-218, 1989; Harrison et al., J. Pediatr. Orthop. 4: 579-584, 1984). It has also been shown that PEMFs (Mooney, Spine 15: 708-712, 1990) and capacitive coupling (Goodwin, Brighton et al., Spine 24: 1349-1356, 1999) can significantly increase the success rate of lumbar fusions. There are also reports of augmentation of peripheral nerve regeneration and function and promotion of angiogenesis (Bassett, Bioessays 6: 36-42, 1987). Patients with persistent rotator cuff tendonitis refractory to steroid injection and other conventional measures, showed significant benefit compared with placebo-treated patients (Binder et al., Lancet 695-698, 1984). Finally, Brighton et al. have shown in rats the ability of an appropriate capacitive coupling electric field to both prevent and reverse vertebral osteoporosis in the lumbar spine (Brighton et al., J. Orthop. Res. 6: 676-684, 1988; Brighton et al., J. Bone Joint Surg. 71: 228-236, 1989).
Research in this area has focused on the effects stimulation has on tissues and cells. For example, it has been conjectured that direct currents do not penetrate cellular membranes, and that control is achieved via extracellular matrix differentiation (Grodzinsky, Crit. Rev. Biomed. Eng. 9:133-199, 1983). In contrast to direct currents, it has been reported that PEMFs can penetrate cell membranes and either stimulate them or directly affect intracellular organelles. An examination of the effect of PEMFs on extracellular matrices and in vivo endochondral ossification found increased synthesis of cartilage molecules and maturation of bone trabeculae (Aaron et al., J. Bone Miner. Res. 4: 227-233, 1989). More recently, Lorich et al. (Clin. Orthop. Related Res. 350: 246-256, 1998) and Brighton et al. (J. Bone Joint Surg. 83-A, 1514-1523, 2001) reported that signal transduction of a capacitively coupled electric signal is via voltage gated calcium channels, whereas signal transduction of PEMFs or combined electromagnetic fields is via the release of calcium from intracellular stores. In all three types of electrical stimulation there is an increase in cytosolic calcium with a subsequent increase in activated (cytoskeletal) calmodulin.
It was reported in 1996 by the present inventors that a cyclic biaxial 0.17% mechanical strain produces a significant increase in TGF-β1 mRNA in cultured MC3T3-E1 bone cells in a Cooper dish (Brighton et al., Biochem. Biophys. Res. Commun. 229: 449-453, 1996). Several significant studies followed in 1997. In one study it was reported that the same cyclic biaxial 0.17% mechanical strain produced a significant increase in PDGF-A mRNA in similar bone cells (Brighton et al., Biochem. Biophys. Res. Commun. 43: 339-346, 1997). It was also reported that a 60 kHz capacitively coupled electric field of 20 mV/cm produced a significant increase in TGF-β1 in similar bone cells in a Cooper dish (Brighton et al., Biochem. Biophys. Res. Commun. 237: 225-229, 1997). It has also been reported that chondrocyte matrix genes and proteins can be up-regulated by specific and selective electric fields (Wang, W., Wang, Z., Zhang, G., Clark, C. C., and Brighton, C. T., Clin. Orthp. and Related Res., 427S: S163-173, 2004; Brighton, C. T., Wang, W., and Clark, C C, Biochem. Biophys. Res. Commun., 342: 556-561, 2006). Further, it has been shown that the gene expression of bone morphogenetic proteins (BMPs) can also be up-regulated by specific and selective electric fields that differ from the electric fields in various signal aspects from those signals that are specific and selective for articular cartilage (Wang, Z., Clark, C. C. and Brighton, C. T., J. Bone Joint Surg., 88: 1053-1065, 2006).
In the above-referenced parent patent application, entitled Regulation of Genes Via Application of Specific and Selective Electrical and Electromagnetic Signals, methods were disclosed for determining the specific and selective electrical and electromagnetic signals for use in creating fields for regulating target genes of diseased or injured tissues. The present invention builds upon the technique described therein by describing the method of regulating expression of one targeted gene family, namely, fibroblastic growth factor-2 (FGF-2) gene expression, through application of a field generated by a specific and selective electrical and electromagnetic signal, for the treatment of bone diseases and injuries including osteoporosis, osteopenia, osteonecrosis, bone defects, fresh fractures, fractures at risk, delayed union, nonunion, bone defects, and as an adjunct in spinal fusion.